Process for the production of low-chromium protein hydrolyzates

ABSTRACT

Described is the chemical hydrolysis of partial hydrolysates obtained by the enzymatic decomposition, in aqueous conditions in the presence of alkaline-earth oxides or hydroxides, of protein-containing raw materials containing chromium. This enables protein hydrolysates with a particularly low chromium content to be obtained which are stable to turbidity during storage. The chemical hydrolysis must be carried out in the presence of compounds selected from the alkaline-earth oxides and hydroxides, and within the critical pH range of 11 to 13.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a process for the production of low-chromiumprotein hydrolyzates.

2. Statement of Related Art

The fate of chromium shavings, which accumulate as waste products in theproduction of chrome-tanned leather, is a growing problem in the leatherindustry. The reason for this is that the presence of the chromium wastein dumps involves the risk of contamination of the ground water byCr(III) and--after possible oxidation--even by Cr(VI). According to dataprovided by Taylor et al., 54,000 tonnes of chromium shavings accumulateannually in the USA alone (J. Am. Leather Chem. Assoc. 1990, 85, 264).

Boiling with alkali, usually lime or magnesium oxide, is one of theoldest methods for detanning chrome leather waste, cf. for example U.S.Pat. No. 4,100,154. According to the teaching of this patent, thealkaline hydrolysis of chrome leather is carried out at temperaturesabove about 90° C. in the presence of calcium oxide or calciumhydroxide. By increasing the reaction temperature, which can be achievedfor example by carrying out the reaction in a pressure reactor, thehydrolytic degradation rate can be further increased. According to thisdocument, chromium is removed to residues "below 5 ppm", although theexact concentrations of collagen hydrolyzate in the solutions and hencethe reference value for the Cr content are not disclosed.

DE-A-1 000 388 describes a process for the production of proteindegradation products in which the hydrolysis of chrome leather iscarried out with water or aqueous ammonia at elevated temperature andpressure.

The desire to avoid the drastic reaction conditions of the conventionalprotein hydrolysis processes mentioned above has resulted in thedevelopment of enzymatic hydrolysis processes. For example, Germanpatent application DE 22 52 281 describes a process for the hydrolysisof skin fragments using neutral or alkaline proteases which are capableof decomposing collagen. Leather fragments inter alia may be used as theskin fragments. Before the enzymatic treatment, the protein of the skinfragment is denatured by heating with water. The enzymatic hydrolysis issubsequently carried out at moderate temperatures of 20° to 70° C. andis typically followed by thermal denaturing of the enzyme and byfiltration of the crude product obtained.

In a more recent publication, Heideman et al. discuss in detail possiblemethods for treating chromium shavings. So far as the hydrolysis ofchromium shavings with proteases is concerned, they come to theconclusion that the preliminary denaturing of the shavings plays acritical role. They obtained optimal results when they first digestedchromium shavings with 10% of lime for about 20 minutes and then treatedthe shavings with protease (cf. Das Leder 1991, 42, 133-143).

It has only recently been shown by Taylor et al. that preliminarydenaturing is not absolutely essential in the enzymatic hydrolysis ofchrome leather waste. In their method, the protein present in the wasteis treated with special mixtures of alkali metal and alkaline earthmetal compounds and alkalases at moderate temperatures withoutpreliminary denaturing. The authors reported on their method at the 85thAnnual Conference of the ALCA (J. Am. Leather Chem. Soc. 1990, 85, 262).In addition, this method is the subject of U.S. Pat. No. 5,094,946.Furthermore, the effectiveness of the method was confirmed in practicaltests carried out at the Danish tannery Svendborg Fingarveri ("BioTimes" [quarterly magazine of the Novo Nordisk Company] 1990, 5, No. 1,pages 4-5). However, the method developed by Taylor et al. has thedisadvantage that the aqueous concentrates thus prepared are not stablein storage, but instead become cloudy over a period of time.

DESCRIPTION OF THE INVENTION

Accordingly, the problem addressed by the present invention was toprovide a process for the production of low-chromium proteinhydrolyzates which would lead to clear, non-clouding products. Anotherproblem addressed by the present invention was to provide a process forthe production of protein hydrolyzates with a lower chromium content bycomparison with the relevant prior art which is important in particularfor the use of these products in cosmetics and in the food industry.

According to the invention, the solution to this problem ischaracterized in that chromium-containing protein-containing rawmaterials are first partly hydrolyzed in aqueous medium in the presenceof an additive from the group of alkaline earth metal oxides orhydroxides and in the presence of proteolytic enzymes and the partialhydrolyzates obtained are subjected to chemical hydrolysis in thepresence of alkaline earth metal oxides or hydroxides at a pH value inthe range from 11 to 13.

Accordingly, the present invention relates to a process for theproduction of low-chromium protein hydrolyzates by hydrolysis ofchromium-containing protein-containing raw materials, in which thechromium-containing protein-containing raw materials are first partlyhydrolyzed in aqueous medium in the presence of an additive from thegroup of alkaline earth metal oxides or hydroxides and in the presenceof proteolytic enzymes and the partial hydrolyzates obtained are thensubjected to chemical hydrolysis--optionally after separation from theinsoluble chromium salts formed by methods known per se--in the presenceof alkali metal compounds from the group of alkaline earth metal oxidesor hydroxides at a pH value of 11 to 13.

The process according to the invention has the advantage that, even inthe form of concentrates with an active substance content of more than40% by weight, the products obtained are stable against clouding instorage and, in addition, are distinguished by an extremely low chromiumcontent.

The first step of the process according to the invention is carried outin known manner.

In a preferred embodiment of the present invention, the first step, i.e.the partial hydrolysis in the presence of proteolytic enzymes, iscarried out without preliminary denaturing of the chromium-containingprotein-containing raw material. This preferred embodiment essentiallycomprises

a) initially introducing the raw material in aqueous medium,

b) introducing an alkaline earth metal oxide or hydroxide as additive toestablish the required pH value and to provide a co-factor for theenzyme,

c) carrying out the reaction in the presence of a proteolytic enzyme ata temperature and a pH value which correspond to the optimum temperatureand optimum pH of the protease used, typically at temperatures of 60° to75° C. and at pH values of 10 to 11.

Reference is specifically made in this regard to U.S. Pat. No.5,094,946. It may be desirable subsequently to separate the crudeproduct obtained in the enzymatic hydrolysis into a partial hydrolyzateand a chromium-containing residue by a separation process such assedimentation, centrifugation or filtration.

It has been found that, in the interests of process economy, "pHstattechnique" is particularly suitable for establishing the pH value inthe first process step. In pH-stat technique, the fall in the pH valuebrought about by protons released during the enzymatic hydrolysis iscounteracted by the continuous introduction of bases so that the pH iskept at a constant level (cf., for example, Jens Adler-Nissen,"Enyzymatic Hydrolysis of Food Proteins", Elsevier, London 1986). Wherethe pH-stat technique in question is applied, it is advisable to carryout the process in the vicinity of the pH-dependent optimum effect ofthe particular enzyme used.

The second step of the process according to the invention, i.e. thechemical hydrolysis, must be carried out in the presence of compoundsfrom the group of alkaline earth metal oxides or hydroxides at a pHvalue of 11 to 13. Lower pH values only promote incomplete hydrolysis sothat, on the one hand, the stability of the protein hydrolyzates toclouding and, on the other hand, the low chromium contents required forthe aqueous protein hydrolyzate are not guaranteed. At higher pH values,the amphoteric chromium(III) hydroxide is in danger of redissolving (cf.Hollemann-Wiberg, "Lehrbuch der anorganischen Chemie", 81-90th Edition,page 876).

Basically, any chromium-containing protein-containing substances may beused as raw materials in the process according to the invention.However, the chrome leather waste accumulating during leather productionin the tanneries is preferably used. By virtue of their particle size inthe mm range, chromium shavings are particularly preferred. Accordingly,chrome leather waste of large surface area, for example leather rags,are best mechanically size-reduced before application of the processaccording to the invention.

The chemical hydrolysis which follows the enzymatic hydrolysis ispreferably carried out at temperatures of 80° to 100° C. and morepreferably at temperatures of 90° to 95° C. The duration of the chemicalhydrolysis should be no less than 5 minutes, the upper limit being 60minutes. Calcium hydroxide or calcium oxide is preferably used as thebase during the chemical hydrolysis.

The chemical hydrolysis step does not require any special technologicalmeasures. Accordingly, it may be carried out in any mixer-equippedreactors. A tanning mixer is mentioned as an example of a suitable openreactor.

The crude product obtained after the chemical hydrolysis step is workedup in known manner. Working up generally comprises

a) one or more filtration steps largely intended to remove the chromiumhydroxide formed. For viscosity reasons, filtration is generally carriedout at temperatures of 60° to 100° C. and, more particularly, attemperatures of 90° to 95° C.;

b) the precipitation of calcium ions, for example by addition of oxalicacid, and subsequent separation of the calcium oxalate formed, forexample by filtration, or by addition of sodium carbonate and separationof the calcium carbonate precipitated;

c) concentration of the protein hydrolyzate to the requiredconcentration.

The products obtained by the process according to the invention areparticularly suitable for use in cosmetics and foods.

The following Examples are intended to illustrate the invention withoutlimiting it in any way.

EXAMPLES

1. General

In Table 1, the Examples according to the invention are identified as E1to E3 while the Comparison Examples are identified as C1 to C2.

2. Examples

2.1. Description of the process according to the invention

2.1.1. Example E1

(a) Enzymatic hydrolysis

300 g of chromium shavings were suspended in 900 g of water at 60° C.The pH value was then adjusted to 9.5-10.5 by addition of calciumhydroxide. 0.6 g of a commercial alkaline protease was then added andthe mixture was kept for 1 hour at 55° to 60° C. During this hydrolysis,the pH value was kept at 9.5 to 10.5 by addition of calcium hydroxide.

(b) Deactivation of the enzyme/filtration

The mixture was then heated to 100° C. and kept at that temperature for15 minutes in order to deactivate the enzyme. To separate chromiumhydroxide and any undissolved calcium hydroxide, the mixture wasfiltered at 95° C.

(c) Chemical hydrolysis

The pH value was increased to 11.0 by addition of more calciumhydroxide, followed by heating for 30 minutes to 90° C. in order furtherto hydrolyze the relatively high molecular weight protein hydrolyzatefragments obtained in the first stage. Undissolved calcium hydroxide andprecipitated chromium oxide were then separated off by filtration. Theprotein hydrolyzate thus obtained was then analyzed for its chromiumcontent in this dilute solution by atomic absorption spectroscopy. Theresults obtained (in μg/ml) can be found in Table 1.

(d) Further working up

In the filtrate, dissolved calcium was precipitated as calcium oxalateand filtered off. The protein hydrolyzate solutions were then adjustedto concentrations of 40% by weight and 50% by weight by distilling offwater.

2.1.2. Example E2

The procedure was as in Example E1 except that, in step (c), the pHvalue was adjusted to 11.5 by addition of calcium hydroxide.

2.1.3. Example E3

The procedure was as in Example E1 except that, in step (c), the pHvalue was adjusted to 12.5 by addition of calcium hydroxide.

2.2. Description of the comparison tests

2.2.1. Comparison Example C1

The procedure was as described in 2.1.1. except that step (c), i.e. thechemical hydrolysis, was left out.

2.2.2. Comparison Example C2

The procedure was as in Example E1 except that, in step (c), the pHvalue as adjusted to 10.5 by addition of calcium hydroxide.

3. Evaluation of the tests

The protein hydrolyzates obtained (E1 to E3 and C1 and C2) were analyzedfor their chromium content and their storage behavior. The resultsobtained are set out in Table 1. The chromium contents are expressed inμg/ml ("ppm") and are based on the aqueous product mixture.

As can be seen from Table 1, the products obtained by the processaccording to the invention (E1 to E3) have distinctly lower chromiumcontents than the products of the pure enzymatic hydrolysis (C1). Inaddition, the products produced by the process according to theinvention remained clear in storage.

The fact that the pH value prevailing during the chemical hydrolysis iscritical is clearly apparent from Example C2 from which it can be seenthat excessively low pH values cause a significantly higher chromiumcontent.

                  TABLE 1                                                         ______________________________________                                                                        Appearance of                                                                 concentrated                                        Chemical hydrolysis                                                                            Chromium product.sup.b                                                        Dura- content.sup.a                                                                        Immedi-                                                                              After                              Example                                                                             Yes/no  pH value tion  (μg/ml)                                                                           ately  3 days                             ______________________________________                                        E1    Yes     11.0     30    <0.2   Clear  Clear                                                     mins.                                                  E2    Yes     11.5     30    <0.1.sup.c                                                                           Clear  Clear                                                     mins.                                                  E3    Yes     12.5     30    <0.1.sup.c                                                                           Clear  Clear                                                     mins.                                                  C1    No      --       --    2.1    Clear  Cloudy                             C2    Yes     10.5     30    0.8    Clear  Clear                                                     mins.                                                  ______________________________________                                         .sup.a Based on the dilute aqueous product mixture; average value of thre     measurements                                                                  .sup.b The remarks in this column apply equally to protein hydrolyzate        concentrates with active substance contents of 40% by weight and 50% by       weight.                                                                       .sup.c In the analysis method selected (atomic absorption spectroscopy),      the detection limit was 0.1 μg/ml, i.e. the figure "<0.1" means that       the chromium content was below the detection limit.                      

What is claimed is:
 1. A process for the production of a low-chromiumprotein hydrolyzate comprising the steps of: (1) partially hydrolyzing achromium-containing nonpartially-denatured protein in an aqueous mediumin the presence of an alkalino earth metal oxide or hydroxide and in thepresence of a proteolytic enzyme to form a partial hydrolyzate; and (2)further hydrolyzing said hydrolyzate in the presence of an alkalineearth metal oxide or hydroxide at a pH value of from about 11 to about13.
 2. The process of claim 1 wherein said chromium-containing proteinis a chrome leather waste from leather production.
 3. The process ofclaim 1 further comprising the step of filtering said partialhydrolyzate at temperature of from about 80° to about 100° C. to removechromium hydroxide before carrying out step (2).
 4. The process of claim3 wherein said temperature is from about 90° to about 95° C.
 5. Theprocess of claim 10 wherein step (2) is carried out at a temperature offrom about of 80° to about 100° C.
 6. The process of claim 5 whereinsaid temperature is from about 90° to about 95° C.
 7. The process ofclaim 1 wherein step (2) is carried out over a period of from about 5 toabout 60 minutes.
 8. The process of claim 1 wherein said alkaline earthmetal oxide is calcium oxide.
 9. The process of claim 10 wherein saidalkaline earth metal hydroxide is calcium hydroxide.
 10. The process ofclaim 1 wherein said process is carried out in a tanning mixer.
 11. Theprocess of claim 1 wherein following step (1) and before step (2) theproteolytic enzyme is deactivated.
 12. The process of claim 11 whereinthe proteolytic enzyme is deactivated by the use of heat.
 13. Theprocess of claim 1 wherein step (1) is carried out at a pH of from about9.5 to about
 11. 14. The process of claim 13 wherein said pH is fromabout 10 to about
 11. 15. A process for the production of a low-chromiumprotein hydrolyzate comprising the steps ofA) partially hydrolyzing achromium-containing nonpartially-denatured protein in an aqueous mediumin the presence of an alkaline earth metal oxide or hydroxide and in thepresence of a proteolytic enzyme at a pH in the range of from about 9.5to about 11 to form a partial hydrolyzate; B) heating the partialhydrolyzate to deactivate the enzyme; C) filtering the partialhydrolyzate at a temperature of from about 80° to about 100° C. toremove insoluble chromium compounds; and D) further hydrolyzing thepartial hydrolyzate in the presence of an alkaline earth metal oxide orhydroxide at a pH of from about 11 to about
 13. 16. The process of claim15 wherein step D) is carried out at a temperature of from about 80° toabout 100° C. for a period of from about 5 to about 60 minutes.
 17. Theprocess of claim 16 wherein said temperature is from about 90° to about95° C.
 18. The process of claim 15 wherein step A) is carried out at atemperature of from about 60° to about 75° C.